5端核苷酸水解酶在所有毒蛇中具有水解核甘單磷酸形成腺苷和磷酸的高分子量的醣蛋白，從結構和功能比對發現和人類的CD73有相似性猜測5端核苷酸在抑制免疫系統中扮演特定角色，然而在不同毒蛇5端核苷酸的N端糖基化與結構以及功能並沒有了解清楚。在我們研究中，比較5端核苷酸的比例與活性在三種眼鏡蛇和兩種出血性的毒蛇，發現台灣眼鏡蛇含0.23%的5端水解酶而且活性最高，利用質譜和X繞射鑑定出台灣眼鏡蛇5端核苷酸上的醣蛋白，發現有四個N端的糖(N311, N333, N347和N518)，之後使用切醣試劑移除5端水解酶醣並且探討酵素活性，發現親和力有明顯的影響，另外，找到有效抑制劑AMPCP可抑制5端水解酶的活性，基於這些研究中，我們推測這個抑制劑減少5端水解酶活性，也許可幫助被蛇毒藥咬傷的受害者，爾後可當作相關藥物治療。

V5NTD, or known as 5’-nucleotidase, is a high molecular weight glycoprotein found in venom of all snake species and has the ability to hydrolyze extracellular AMP (adenosine monophosphate) to adenosine and Pi. It presumably plays a role in inhibiting immune response similar to human V5NTD (CD73) as they share similar overall protein fold with the same 3D structure at the active site. The relative amount of V5NTD in different snake venoms and the structure and function of its N-glycosylation, however, are not well understood. In this study, we first compare the venom content and activity of V5NTD from three cobra species of N. atra, N. kaouthia and N. nivea and two viper species of T. mucrosquamatus and D. acutus. The highest enzymatic activity is found in the crude venom of N. atra with the presence of ~ 0.23% w/w of V5NTD. We then apply both MASS spectrometry and X-ray diffraction method to identify the four glycan structures of V5NTD from N. atra and study their effect on its enzyme kinetics by treating with Endo F3. The results indicate that the N-glycan structures at N311, N333, N347 and N518 are mainly of complex type and exhibit different degree of structural dynamics. Removing all N-glycans from V5NTD has delicate, but small, effect on its enzyme kinetics. We also find that V5NTD can be effectively inhibited by AMPCP (adenosine 5’(-methylene) diphosphate) at sub-mM concentration range. Based on these observations, we suggest that the inhibition of V5NTD activity at the bitten tissue area may help the wound healing process of the victims suffering from snakebite.

Chapter 1 Introduction
1.1 Snake venom proteomes………………………………………………………...............…..3
1.2 5’-nucleotidase in Naja atra (Taiwan cobra)…………………………………........…..4
1.3 N-link glycosylation…...................................................................................5
1.4 Protein purification……………………………………………………………….....................6
1.5 Enzyme kinetics ………………………………………………………………....................…..7
Chapter 2 Materials and Methods
2.1 Materials………………………………………………………………………...........................…14
2.2 Compare total amount of 5’-nucleotidase in snake venom…………………...….14
2.3 Crude venom assay with AMP…………………………………………………...................15
2.4 Purification of 5’-nucleotidase from Taiwan cobra (Naja atra) venom………..15
2.5 5’-nucleotidase crystallization…………………………………………………................…17
2.6 5’-nucleotidase data collection, structure determination and refinement…..17
2.7 5’-nucleotidase kinetic assay with AMP….…………………………….......................18
2.8 5’-nucleotidase inhibition assay………………………………………………................….18
2.9 DeN-glycosylated of V5NTD hydrolyzing AMP………………………………..............19
2.10 Identification of V5NTD N-glycosylation sites……………………………….............19
Chapter 3 Results
3.1 Identification of the HMW from snake venom………………………………….............20
3.2 Snake venom activity with AMP…………………………………………………..................20
3.3 Purification of the V5NTD from Naja atra…….......………………………..................21
3.4 V5NTD crystal and data collection……………………………………………................…22
3.5 V5NTD enzyme by using AMP as a substrate……………………………….............…23
3.6 V5NTD inhibition with AMPCP……………………………………………….....................…23
3.7 DeN-glycosylated of V5NTD activity with AMP……………………………...........…...24
3.8 V5NTD N-glycosylation sites….……….............…………………………..................….24
Chapter 4 Discussion……………………………………………………….......................………...50
Conclusion……………………………………………………………………............................……....60
Reference…………………………………………………………………….............................………..61

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